5H-Pyrano[2,3-d: 6,5-d&#39;]dipyrimidine derivatives having an antibacterial, antiviral and immuno-modulating activity

ABSTRACT

The present invention relates to novel compounds having the formula                    
     The compounds have antibacterial, antiviral, and immuno-modulating activity.

FIELD OF THE INVENTION

The invention relates to the area of organic chemistry and medicine,specifically to barbituric acids and their derivatives, and is designedfor use as substances having antiviral, antichlamydic, immunomodulatingand antitumoral activity.

DESCRIPTION OF PRIOR ART

Many derivatives of barbituric acid are biologically active substances.The derivatives of pyrimidine are nucleic bases (uracil, thymine,cytosine), vitamins (thiamine, phosphothiamine), coenzymes(cocarboxylase), and they are utilized as pharmaceuticals havingsoporific, anticonvulsive (barbiturates, hexamidine, benzonal), diuretic(mercusal), antiinflammatory (pentoxil, methyluracil), and antithyroidal(methylthiouracil) actions; they are synthetic analogs of vitamins(“bephothiamine”), anabolic (orotic acid), anti-inflammatory andantibacterial (sulfazin, sulfadimethazin, sulfamonomethoxin,sulfadimethoxin, bactrim, salazodimethoxin), antimalarial (chloridin),anticarcinogenic (dopan, phosphamide, ethimidide, fluorouracil,fluorofur, cytarabin) substances [1, 2].

In recent decades, systems in which the pyrimidine ring is condensedwith other heterocycles have received considerable attention. Suchheterocycles are frequently analogs of natural, biologically activesubstances. They include purines, which occur in natural and syntheticbiologically active substances: nucleic acids (adenine, guanine), ATP,substances that excite the central nervous system (caffeine,theobromine, theophylline, “nihexine,” diprophylline, xanthinolnictoninate), substances used in suppressing tissue incompatibility inorgan transplants (azathioprine) (cytostatic and immumodepressiveeffect), and substances having an anabolic (inosine) or antileukemiceffect (mercaptopurine), pyrazolo[3,4-d]pyrimidines used to treatdiseases accompanied by hyperuremia (allopurinol); pteridines, used asdiuretics (triamterene), vitamins (riboflavin, folic acid),anticarcinogenic drugs (methotrexate); pyrimido[5,4-d]pyrimidines havingvasodilational properties (dipyridamole [1, 2].

Data on the biological activity of the most varied derivatives of5-ylidenebarbituric acids have been summarized in a review [3] whichnotes the anticoma, antimicrobial, spasmolytic, antipyretic andantitumor activity of these substances. Data has also been obtained onthe biological activity of certain 5-arylidenebarbituric acids [4-11],5-aminomethylenebarbituric acids [12], products of the reaction ofbarbituric acids with isocyanates, isothiocynates -5-arylcarbamoylbarbituric acids [13-16], pyrazolo[3,4-d]pyrimidinesobtained by the condensation of 6-hydrainouracils with iso(thio)cyanates[17], 5-deazaflavins [18-20], condensation products of 6-aminouracilswith nitrosobenzene-10-alkyl(halogenophenyl)-3-methylflavins [21, 22],derivatives of pyrrolo[2,3-d]pyrimidines [23],7-methyl-5-hexyl-1H-pyrazolo-[3,4-d]-pyrimidino-4,6(5H,7H)-dion obtainedby the action of Wilsmeyer's reagent on the corresponding6-hydrazinouracil [24], pyrano[2,3-d]pyrimidines [25-27],5-(3-nitrophenyl)-4-oxo-2-thioxo-1,3,7-triphenyl-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidines[28-33],pyrimido[5,4-e][1,2,4]triazino-5,7(1H,6H)-dions [34],5-dialkylaminomethylpyridines [35, 36], and pyrimido[4,5-c] pyridazines[37]. The foregoing compounds have pesticidal, antitumor, antimicrobial,immunosuppressive, nootropic and antihypertensive and antiallergicactivity.

Only a few examples are known of the formation of the pyrano[2,3-d]6,5-d′]dipyridimine system by the interaction of barbituric acids with3-acylchromones [38, 39]. There is no information as to their biologicalactivity. At the same time, available information on the biologicalactivity of 5-substituted barbituric acids and condensed systemscontaining the pyrimidine ion fragment, as stated above, indicates thatthey have varying degrees of biological activity. The effectiveness ofmany of the substances that have been studied, however, is notsatisfactorily high, and many of them are toxic and cause side-effects.Furthermore, bacteria, viruses and tumor cells rapidly developresistance to the existing drugs, making their employment inefficient[40-46].

The material above suggests the potential usefulness of conducting ascientific search in the area of the synthesis of effective newbiological substances by condensing barbituric acids with carbonylcompounds, producing, specifically, derivatives ofpyrano[2,3-d:6,5-d′]dipyrimidine. The prototype of the invention, i.e.,the substance closest in chemical nature to the claimed substance, is1,3,7,9-tetramethyl-5-(3-chromonyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidino-2,4,6,8(1H,3H,7H,9H)-tetraon[38]. It is obtained by heating 1,3-dimethylbarbituric acid with3-chromoncarbaldehyde, first in a mixture with pyridine andtriethylamine, and then in acetic acid containing sulfur. As notedabove, available sources contain no information on its biologicalactivity.

OBJECT OF THE INVENTION

The object of the invention is to create new substances havingantimicrobial, antiviral immunomodulating and antitumor activities.

ESSENCE OF THE INVENTION

The object of the invention is achieved by the synthesis of novelcompounds—derivatives of pyrano[2,3-d:6,5-d′]dipyrimidine of generalformula:

where:

R¹ is selected from the group HYDROXY GROUP, MERCAPTO GROUP, HALOGEN,

R² is selected from the group HYDROXY GROUP, ALKOXY GROUP, HALOGEN,

R³ is selected from the group HYDROGEN, ARYL.

The best variants of the claimed substances are when

R¹ = R² = OH, R³ = 4-O₂NC₆H₄ (I), R¹ = R² = Cl, R³ = C₆H₅ (II), R¹ = R²= OH, R³ = 4-IC₅H₄ (III), R¹ = R² = OH, R³ = H (IV), R¹ = OH, R² = OCH₃,R³ = 4-O₂NC₆H₄ (V), R¹ = R² = OH, R³ = 4-ClC₆H₄ (VI), R¹ = R² = OH, R³ =4-BrC₆H₄ (VII), R¹ = SH, R² = OH, R³ = 4-ClC₆H₄ (VIII), R¹ = SH, R² =OH, R³ = 4-O₂NC₆H₄ (IX).

It should be noted at once that the use of other representatives of thealkoxy group, the mercapto group, halogens and aryls does not differ inprincipal in the synthesis process or in the biological activities ofthe substances obtained, i.e., it is not the specific elements in theR¹, R² and R³ radicals that are of importance, but that they belong tothe groups cited in the general formula.

The proposed best variants are listed in the table that follows forclarity.

Variant R¹ R² R³ I OH OH 4-O₂NC₆H₄ II Cl Cl C₆H₅ III OH OH 4-IC₅H₄ IV OHOH H V OH OCH₃ 4-O₂NC₆H₄ VI OH OH 4-ClC₆H₄ VII OH OH 4-BrC₆H₄ VIII SH OH4-ClC₆H₄ IX SH OH 4-O₂NC₆H₄

The general formula of the claimed substances and all of the variants ofspecific compounds listed above are new, and are not known to us fromavailable sources of information. Furthermore, their synthesis and theexistence of marked biological activity in them does not followobviously from the current level of technology, i.e., they are notobvious to one skilled in the art.

DISCLOSURE OF THE INVENTION

The essence of the invention is explained below by five general examplesof the synthesis of the claimed substances, the three best claims forsynthesis variants known to the inventors as of the date of delivery ofthe application, three summary tables of the chemico-physicalcharacteristics of the claimed substances giving their yields, meltingpoints, as well as the results of four series of experiments todetermine the comparative medico-biological properties of the claimedsubstances (with corresponding Tables of Quantitative Measurements),where:

EXAMPLE 1 Variant of the Synthesis of5-Aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d:6,5-d′]dipyrimidines (I,III, IV, VI, VII) and5-aryl-4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d′]dipyrimidines(VIII, IX) EXAMPLE 2 Variant of the Synthesis of5-Aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d:6,5-d′]dipyrimidines (I,III, IV, VI, VII) and5-aryl4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d′]dipyrimidines(VIII, IX) EXAMPLE 3 Variant of the Synthesis of2,4,6,8-Tetrahydroxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine(I) EXAMPLE 4 Variant of the Synthesis of5P-Phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (II)EXAMPLE 5 Variant of the Synthesis of2,8-Dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine(V) EXAMPLE 6 Best Variant Known to the Inventors for the Synthesis of4-6-Dihydroxy-2,8-dimercapto-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine(IX) EXAMPLE 7 Best Variant Known to the Inventors for the Synthesis of5-Phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (II)EXAMPLE 8 Best Variant Known to the Inventors for the Synthesis of2,8-Dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (V)

Table 1—PMR spectra of solutions of claimed substances—derivatives of5H-pyrano[2,3-d:6,5-d′]dipyrimidines—in DMSO-d₆ (δ, ppm)

Table 2—¹³C NMR spectra of solutions of claimed substances—derivativesof 5H-pyrano[2,3-d:6,5-d′]dipyrimidines—in DMSO-d₆ (δ, ppm)

Table 3—yields, temperatures of decomposition and elemental analysis ofclaimed substances—derivatives of 5H-pyrano[2,3-d:6,5-d′]dipyrimidines

Experiment 1 (with Table 4)—determination of the activity of claimedcompounds on herpes simplex virus

Experiment 2 (with Table 5)—determination of the interferon-inducingactivity of claimed compounds.

Experiment 3 with (Table 6)—determination of activity of claimedcompounds on C. trachomatis.

Experiment 4 (with Table 7)—determination of acute toxicity of claimedcompounds.

EXAMPLE 1 Variant of the Synthesis of5-Aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d:6,5-d′]dipyrimidines (I,III, IV, VI, VII) and5-aryl4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d′]dipyrimidines(VIII, IX)

A mixture of 3 mmol of the pyridine salt of5,5′-arylidenebis(2-thio)barbituric acid and 55 mmol of POCl₃ is heatedwith stirring to boiling. After the precipitate is dissolved (after˜1hour), 3.5 mmol of P₂O₅ is added to the mixture. The boiling mixture isstirred for 5-7 hours, and then poured into 150-200 ml of cold water andleft over night. The precipitate that comes out of solution is filtered,washed with ethanol or acetone, recrystallized from water. Yield 20-30%.

EXAMPLE 2 Variant of the Synthesis of5-Aryl-2,4,6,8-tetrahydroxy-5H-pyrano[2,3-d:6,5-d′]dipyrimidines (I,III, IV, VI, VII) and5-aryl-4,6-dihydroxy-2,8-dimercapto-5H-pyrano[2,3-d:6,5-d′]dipyrimidines(VIII, IX)

A mixture of 0.04 mol of the pyridine salt of5,5′-arylidenebis(2-thio)barbituric acid, 0.035 mol of POCl₃, 0.02 molof P₂O₅, and 7-10 ml of chloroform are stirred while being boiled for 4hours. The reaction mixture is poured into 100 ml of cold water and leftover night. Separation and purification of the resulting substances isas described above. Yield 23-86%.

EXAMPLE 3 Variant of the Synthesis of2,4,6,8-Tetrahydroxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine(I)

To a mixture of 3.4 mmol of2,8-dihydroxy4,6-dimethoxy-5-(p-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine,10 ml of acetic acid, and 10 ml of acetic anhydride there are added 8-12drops of concentrated sulfuric acid. The mixture is stirred while beingboiled for 8-10 hours, and then poured into 150 ml of cold water andleft over night. Separation and purification of the resulting substancesis as described above. Yield 25-30%

EXAMPLE 4 Variant of the Synthesis of5P-Phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (II)

A mixture of 3 mmol of the pyridine salt of5,5′-benzylidenebisbarbituric acid and 55 mmol of POCl₃ is heated withstirring to boiling. After the precipitate dissolves (about 1 hour), 3.5mmol of P₂O₅ are added to the solution. The boiling mixture is stirredfor 4 hours and then poured into 150-200 ml of cold water and left overnight. The precipitate that comes out of solution is filtered and washedwith ethanol or acetone. Yield 10%.

EXAMPLE 5 Variant of the Synthesis of2,8-Dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (V)

A mixture of 14 mmol of 6-methoxyuracil, 7 mmol of p-nitrobenzaldehyde,10 ml of acetic acid and 10 ml of acetic anhydride are stirred at110-115° C. for 3-5 h. The precipitate that comes out of solution isfiltered, washed with ethanol and dried.

Examples of the Best Variants of the Synthesis of the Claimed Substancesof Formula (I) Known to the Inventors.

EXAMPLE 6 Best Variant for the Synthesis of4-6-Dihydroxy-2,8-dimercapto-5-(p-nitrophenyl)-5[-pyrano[2,3-d:6,5-d:′]dipyrimidine(IX)

A mixture of 0.04 mmol of the pyridine salt of5,5′-(p-nitrobenzylidene)bis(2-thiobarbituric) acid, 0.035 mol of POCl₃,0.02 mol of P₂O₅ and 7-10 ml of chloroform are stirred while boiling for4 hours. Then the reaction mixture is poured into 100 ml of cold waterand left over night. Separation and purification of the resultingsubstances are as described above. Yield 85%.

EXAMPLE 7 Best Variant Known to the Inventors for the Synthesis of5-Phenyl-2,4,6,8-tetrachloro-5H-pyrano[2,3-d:6,5-d′]dipyrimidine (II)

A mixture of 3 mmol of the pyridine acid of5,5′-benzylidenebisbarbituric acid and 55 mmol of POCl₃ is heated withstirring to boiling. After the precipitate dissolves (about 1 hour), 3.5mmol of P₂O₅ are added to the solution. The boiling mixture is stirredfor 4 hours and then poured into 150-200 ml of cold water and left overnight. The precipitate that comes out of solution is filtered and washedwith ethanol or acetone. Yield 10%.

EXAMPLE 8 Best Variant of the Synthesis of2,8-Dihydroxy-4,6-dimethoxy-5-(4-nitrophenyl)-5H-pyrano[2,3-d:6.5-d′]dipyrimidine(V)

A mixture of 14 mmol of 6-methoxyuracil, 7 mmol of p-nitrobenzaldehyde,10 ml of acetic acid and 10 ml of acetic anhydride are stirred at110-115° C. for 3-5 h. The precipitate that comes out of solution isfiltered, washed with ethanol and dried. Yield 46%.

TABLE 1 PMR spectra of solutions of claimed substances — derivatives of5H-pyrano[2,3-d:6,5-d′] dipyrimidines — in DMSO-d₆ (δ, ppm) No C⁵ Ar NHI 4.62 s 7.60 d (2H, H^(2.6)), 8.08 d (2H, H^(3.5)), J 8.4 Hz 11.18 s,12.35 narr. s. II 5.43 s 7.32 M (3H), 7.40 d (2H, J 7.4 Hz) — III 4.45 s7.10 d (2H, H^(2.6)), 7.57 d (2H, H^(3.5)), J 8.1 Hz 11.14 s, 12.20narr. s. IV 2.86 s — 11.19 s, 12.06 V VI 4.48 s 7.26 d (2H, H^(2.6)),7.31 d (2H, H^(3.5)), J 8.3 Hz 11.16 s, 12.30 narr. s. VII 4.48 s 7.25 d(2H, H^(2.6)), 7.40 d (2H, H^(3.5)), J 8.2 Hz 11.14 s, 12.30 narr. s.VIII 4.51 s 7.27 d (2H, H^(2.6)), 7.34 d (2H, H^(3.5)), J 8.4 Hz 12.52 sIX 4.84 s 7.62 d (2H, H^(2.6)), 8.09 d (2H, H^(3.5)), J 8.4 Hz 12.53 s

TABLE 1 PMR spectra of solutions of claimed substances — derivatives of5H-pyrano[2,3-d:6,5-d′] dipyrimidines — in DMSO-d₆ (δ, ppm) No C⁵ Ar NHI 4.62 s 7.60 d (2H, H^(2.6)), 8.08 d (2H, H^(3.5)), J 8.4 Hz 11.18 s,12.35 narr. s. II 5.43 s 7.32 M (3H), 7.40 d (2H, J 7.4 Hz) — III 4.45 s7.10 d (2H, H^(2.6)), 7.57 d (2H, H^(3.5)), J 8.1 Hz 11.14 s, 12.20narr. s. IV 2.86 s — 11.19 s, 12.06 V VI 4.48 s 7.26 d (2H, H^(2.6)),7.31 d (2H, H^(3.5)), J 8.3 Hz 11.16 s, 12.30 narr. s. VII 4.48 s 7.25 d(2H, H^(2.6)), 7.40 d (2H, H^(3.5)), J 8.2 Hz 11.14 s, 12.30 narr. s.VIII 4.51 s 7.27 d (2H, H^(2.6)), 7.34 d (2H, H^(3.5)), J 8.4 Hz 12.52 sIX 4.84 s 7.62 d (2H, H^(2.6)), 8.09 d (2H, H^(3.5)), J 8.4 Hz 12.53 s

TABLE 3 Yields, temperatures of decomposition and elemental analysis ofclaimed substances — derivatives of 5H-pyrano[2,3-d:6,5-d′]dipyrimidines Temp. Yield decomp Found, % Calculated, % No % ° C. C H NFormula C H N I 23 >300 48.26 2.76 18.64 C₁₅H₉N₆O₇ 48.53 2.44 18.86 II10  190 44.78 1.34 14.25 C₁₅H₆Cl₄N₄O 45.04 1.50 14.00 III 33 >300 40.032.31 12.04 C₁₅H₉IN₄O₅ 39.84 2.01 12.36 IV 48 >300 55.13 2.89 17.02C₁₆H₁₀N₄O₅ 55.22 3.09 17.17 V 46  294 51.23 3.37 17.44 C₁₇H₁₃N₅O₇ 51.133.28 17.54 VI 47 >300 48.30 3.24 14.36 C₁₆H₉ClN₄O₅ 49.95 2.51 15.53 VII29  310 44.75 2.15 13.97 C₁₆H₉BrN₄O₅ 44.47 2.24 13.83 VIII 58  265 45.942.23 14.40 C₁₆H₉ClN₄O₃S₂ 45.86 2.31 14.26 IX 86  268 44.32 2.18 17.41C₁₅H₉N₅O₅S₂ 44.66 2.25 17.36

EXPERIMENT SECTION Experiment 1

Determination of the Activity of Claimed Compounds on Herpes SimplexVirus

The antiviral activity was studied relative to the virus herpes simplextype I (VPG-I/Leningrad/248/88) by a standard method [41, 42]. The viruswas grown on a reinoculated Vero cell culture obtained from the cellculture bank of the Institute of Cytology of the Russian Academy ofSciences.

Procedure

To cells grown on medium RPMI-1640 with 10% calf serum and placed in thewells of a 96-well plate, the virus was added to give a finalconcentration of 10 particles/ml and the claimed compounds dissolved inDMSO were added to give a final concentrations of 100, 10, and 1 mg/l.We used 5 individual wells for every concentration tested. The plate wasincubated for 60 min at 38° C. in a CO2 incubator. After incubation thevirus was removed and new medium was added that contained the claimedcompounds in the test concentrations.

The results were evaluated according to the existence of a cytopathicaction of the virus on the cells after 36 hours of incubation at 38° C.in the CO2 incubator.

The following controls were used in the experiment:

1. Control of culture cells (capability for normal growth).

2. Control of virus (assessment of reproductive capability).

3. Control of antiviral activity of an antiviral preparation—acylcovir.

4. Control of compounds (toxicity of compounds).

5. Control of solvent (DMSO) on toxicity.

To access the cytopathic effect of the virus, we counted the number ofunchanged cells in 100 fields formed by a specialized grid on theeyepiece of the inversion microscope. The results are presented in Table4.

TABLE 4 Activity of claimed compounds on herpes simplex virusConcentration of test compounds (mg/l) No. Compound 100 50 10  1Acyclovir —* — 9600** (80%)**  2 DMSO 12000 12000 12000  3 Cell control12000 12000 12000  4 I 9600 (80%) 8400 (70%) 7200 (60%)  5 II CytotoxicCytotoxic —  6 III 4800 (40%) — —  7 IV Cytotoxic — —  8 V 6000 (50%) ——  9 VI 6000 (50%) — — 10 VII 3600 (30%) — — 11 VIII 10800 (90%) 6000(50%) 2400 (20%) 12 IX 4800 (40%) — — *the indicated concentration ofthe drug was not tested, **number of cells in 100 fields, ***the numberin parentheses is the percent protection of the cells against the viruscompared to the cell culture control.

The results presented in Table 4 indicate that all of the claimedcompounds exhibit antiherpes activity comparable to that of the standarddrug acyclovir. The other claimed compounds exhibited less markedactivity in suppressing the reproduction of the virus under theexperimental conditions selected.

Experiment 2

Determination of the Interferon-inducing Activity of Claimed Compounds

Interferon synthesis induction by the claimed preparations was conductedon a primary culture of human lymphocytes (the cells in the human bodyare the main producers of interferon). To obtain a culture of thelymphocytes we used fresh blood (12 hours after collection) from healthyanimals (not from the second group). To isolate the lymphocytes,heparinized blood obtained from a healthy donor was centrifuged in adensity gradient (phycoll-verographin) of 1.71 g/cm³ to isolate thefraction of the immunocompetent cells.

This fraction was taken and diluted with RPMI-1640 nutrient mediumcontaining 5% bovine fetal serum, 0.3 mg/ml of L-glutamine, 100 units/mlof penicillin, and 50 mg/ml of streptomycin. The lymphocyteconcentration was determined after staining with methyl blue and thenumber of cells in the Goryaev chamber was calculated. The startingsolutions of the claimed substances were diluted with RPMI-1640 nutrientmedium to give final concentrations of the substances of 100 mg/l, 10mg/l, and 1 mg/l after the lymphocyte suspension was added. The finalconcentration of the lymphocytes in the induction mixture was 3·10⁶cells/ml. The following controls were processed in parallel with thetest samples:

1) control for the spontaneous production of interferon (IFN) bylymphocytes;

2) control for the process occurring under the action of standardizedINF inducer N-methyl-N-(a,D-glucopyranosyl)ammonium 10-methylenecarboxylate of acridone (cycloferon).

3) control for the process occurring under the action of standardizedINF inducer Neovir with the corresponding content of DMSO in theexperimental samples.

4) control for the spontaneous production of interferon in the presenceof DMSO in a quantity corresponding to the test samples.

The control and the experimental samples were incubated for 24 hours at37° C. Following incubation the samples were centrifuged at 2000 g tosettle out the cellular elements and the INF-containing supernatant wastaken from the samples and analyzed for INF content. The cell residuewas resuspended in the same volume of nutrient medium, stained with avital dye—trypan blue—and the number of cells in the Goryaev chamber wascalculated (as described above) to determine the cytotoxic activity ofthe preparations.

For the quantitative determination of INF in the control andexperimental samples we used the ProCon IF2 Plus immunoenzyme testsystem sold by TOO Proteinovyi Kontur. To determine the quantity ofinterferon s in the sample we used a solid-phase immunoenzyme methodutilizing horseradish peroxidase as the enzyme indicator. The activityof the bound peroxidase was measured using an automated photometer at awavelength of 450 nm for the microplanchets with a microprocessor. Tocalculate the results we determined in parallel the activity of INF instandard solutions of INF containing known amounts of the preparation.On the basis of the results obtained we plotted a calibration curve thatallowed us to obtain data expressed in International Units (IU) from themicroprocessor of the automated photometer. The results of the analysisare expressed in IU of activity of INF per ml in the given inductionsystem containing 3·10⁶ lymphocytes/ml. Every experimental and controlpoint was studied in 4 parallel runs.

Controls of the Immunoenzyme Reaction

1. Control of DMSO with the nutrient medium.

2. Control of the system components (according to Instructions). Allresults were taken into account only if the controls corresponded to thepassport data of the system.

The results obtained were analyzed statistically using the t-test andthe confidence interval at p=0.05 was calculated. Analysis of theconvergence of the results of the parallel tests were performed. As aresult of the experiment it was established that all of the claimedcompounds have an ability to induce INF synthesis entirely comparable tothat of known interferon-inducing substances, which demonstrates theirantiviral and antitumor effectiveness (Table 5).

TABLE 5 Quantitative assessment of IFN-inducing activity of claimedcompounds. Content of INF in induction mixture after 24 hours ofincubation with various concentrations of the drugs. Interferon-inducingactivity of the drug at different concentrations (mg/ml), IU/3 · 10⁴lymp/ml No. Compound 100 mg/ml 10 mg/ml 1 mg/ml  1. Lymphocyte 0 0 0control  2. Cycloferon 58.0 ± 1.4 22.0 ± 2.5 3.8 ± 0.8  3. Neovir 66.0 ±1.4 24.5 ± 1.2 4.1 ± 0.5  4. Poly I/poly C —* 43.6 ± 2.0 10.5 ± 0.8  5.DMSO 0 0 0  6. I 80.0 ± 1.2 28.0 ± 1.5 8.0 ± 0.8  7. II 40.0 ± 1.4 16.0± 0.8 2.0 ± 0.4  8. III 243.2 ± 1.0 112.6 ± 2.0 56.5 ± 1.0  9. IV 74.0 ±1.8 29.9 ± 2.0 3.8 ± 1.5 10. V 108.9 ± 2.0 50.6 ± 1.8 10.5 ± 2.5 11. VI92.2 ± 2.1 36.9 ± 1.2 6.5 ± 1.4 12. VII 22.2 ± 1.4 10.6 ± 1.8 5.6 ± 1.613. VIII 70.5 ± 2.5 23.2 ± 1.5 1.5 ± 1.0 14. IX 64.0 ± 1.8 28.0 ± 2.56.8 ± 2.0 *the preparation was not tested in this concentration

Experiment 3

Determination of Activity of Claimed Compounds on Chlamydia trachomatis

The antimicrobial activity of the claimed compounds was studied on C.trachomatis D323—a standard strain from the collection of the Departmentof Microbiology of the Pavlov St.-Petersburg State University. Thisstrain was isolated from a patient with chlamydic urethritis. It has themorphology and physiological activity characteristic of the species, andis sensitive to the action of drugs used in treating chlamydicinfections.

In the work reported here we used McCoy and L929 cell cultures obtainedfrom the Institute of Cytology of the Russian Academy of Sciences.

Procedure

The cells were grown in flasks of neutral glass in RPMI-1640 medium withan addition of 10% fetal calf serum. The experiment was placed in glass(toxicity-free) flat-bottomed flasks and covered with glass. The cellswere introduced into the medium in a final concentration of 1·10cells/ml. After a monolayer was obtained, standard infectious doses ofchlamydia that had been maintained in a frozen state at −70° C. wereadded to test tubes. At the same time, the test compounds were added tothe cells to give a final concentration of 100 mg/ml. The sample wascentrifuged at 2400 g for 60 min at room temperature and incubated at37° C. for 2 hours. After this, the nutrient medium was replaced by anew medium that contained 5% fetal calf serum and cycloheximide (2μg/ml) and the claimed compounds were readded in the same concentration.A parallel series of samples was run using medium without thecycloheximide in order to avoid its influence on the test substances.The samples were incubated for 48 hours in a CO2 incubator.

The controls included: controls of the cell cultures, control for theaction of solvents, control for the action of chlamydia in the absenceof any preparations at all, control for the sensitivity of chlamydia tostandard antimicrobial preparations—cyprofloxacin and abactal [46],control of the test compounds on toxicity relative to cell cultures.

The results were evaluated from the detection of chlamydia cytoplasmaticinclusion by an immunoenzyme method (MicroTrac Chlamydia trachomatisDirect Specimen Test) and from chlamydia antigens using CylaMonoScreen(Russian-British Joint Venture, 66 Regent's Park Road, London NW 1 7SX)[46]. The effect of the preparations was determined by analyzing thestate of the monolayer and the number of cells with CPE based on acomparison to the control (cell culture infected with C. trachomatisD323) by counting the number of unchanged cells in 100 viewing fieldsproduced by a specialized grid for the microscope eyepiece.

The results of the control samples satisfying the requirements of theexperiment:

control of cell culture—the morphology of the cells and the state of themonolayer are appropriate to the given type of cells, control of growthof chlamydia in the cell culture—the existence of CPE in the monolayer,control of the action of a standard antimicrobial preparation—areduction in the number of cells with CPE in the monolayer compared tothe previous control, control of toxicity of claimed compounds—notoxicity, control for the activity of solvents—no toxic effect on thecells. The results of the tests are presented in Table 5.

TABLE 6 Activity of claimed compounds on C. trachomatis Concentration oftest compounds (mg/l) No. Compound 100 30  1. Cell control 8000* 8000 2. DMSO 8000 8000  3. Control of inf. cells 6400 6400  4. I 7640(80%)** 7520 (70%)  5. II cytotoxic cytotoxic  6. III cytotoxic —  7. IVcytotoxic 7040 (40%)  8. V cytotoxic —  9. VI 6560 (15%) — 10. VIIcytotoxic — 11. VIII 7360 (60%) — 12. IX 7360 (60%) — 13. Cyprofloxacin7200 (50%) 6880 (30%) 14. Abactam 7200 (50%) 7040 (40%) *number of cellsin 100 fields, **the number in parentheses is the percent protection ofthe cells against infection, ***the indicated concentration of the drugwas not tested.

The data obtained demonstrate that the claimed compounds can be used totreat illnesses caused by chlamydia.

Experiment 4 Determination of Acute Toxicity

Acute toxicity was determined on non-linebred white mice weighing 18-20g. Emulsions of certain of the claimed compounds were prepared in arange of concentrations: 1500, 700, 500, 100, 20 and 5 μg/kg. Fiveanimals were used to study each specific concentration of a compound.The preparation was administered once a day by mouth or intravenously.The observation period was 14 days On days 1, 8 and 15 the animals inevery group were weighed. For a control we used animals who received theemulsion without the test compounds.

All animals, both that died in the course of the experiment and thosethat survived to the end of the experiment, were opened to allowmacroscopic examination of internal organs.

In the course of the experiment no loss of weight was observed or anychange in behavior or external appearance, nor was there any loss oflife. Based on the results of the macroscopic examination of internalorgans of the animals in the test and the control groups, nopathological findings were made (Table 7).

TABLE 7 Acute toxicity (LD 50) of some of the claimed compounds No.Compound LD50, mg/kg 1. I 1600 2. VIII 1800

The data show that when the claimed compounds were taken by mouth orintravenously at a dosage level of 1500 mg/kg, no acute toxicity wasfound for mice.

Industrial Application

Examples 1-8 of the practical synthesis and the chemico-physicalanalysis of the claimed compounds listed in Tables 1-3 confirm thepossibility of the laboratory and industrial synthesis of all nineclaimed compounds, which are capable of being manufactured by the modempharmaceutical industry, as well as their precise identification usingstandard control methods.

The series of experiments on identifying the biological propertiespresented in the four reports presented showed that the claimedcompounds exhibit biological activity relative to a variety ofmicroorganisms, including chlamydia, herpes simplex virus, and also haveinterferon-inducing activity. This last suggests the possibility ofusing them to treat herpes, other viral and certain tumor illnesses. Theinformation presented above prove that the goal of the invention wasachieved, namely, the synthesis of a novel class of heterocycliccompounds having a high and broad biological activity, in particular,immunostimulating, antichlamydic and antiviral activities.

Thus, we believe that the claimed compounds (novel substances) satisfyall requirements for a patent: they are novel, non-obvious, and can beproduced on an industrial basis.

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39. . . . gential herpes [spelling correction]

41. . . . gential herpes [spelling correction]

What is claimed is:
 1. A compound having the formula:

where R¹ is selected from the group consisting of hydroxy group,mercapto group, and halogen; R² is selected from the group consisting ofhydroxy group, alkoxy group, and halogen; and R³ selected from the groupconsisting of hydrogen and aryl, optionally substituted by halogen ornitro.
 2. A compound according to claim 1, wherein R¹ is OH, R² is OH,and R³ is 4-O₂NC₆H₄.
 3. A compound according to claim 1, wherein R¹ isCl, R² is Cl, and R³ is C₆H₅.
 4. A compound according to claim 1,wherein R¹ is OH, R² is OH, and R³ is 4-IC₅H₄.
 5. A compound accordingto claim 1, wherein R¹ is OH, R² is OH, and R³ is H.
 6. A compoundaccording to claim 1, wherein R¹ is OH, R² is OCH₃, and R³ is 4-O₂NC₆H₄.
 7. A compound according to claim 1, wherein R¹ is OH, R² isOH, and R³ is 4-ClC₆H₄.
 8. A compound according to claim 1, wherein R¹is OH, R² is OH, and R³ is 4-BrC₆H₄.
 9. A compound according to claim 1,wherein R¹ is SH, R² is OH, and R³ is 4-ClC₆H₄.
 10. A compound accordingto claim 1, wherein R¹ is SH, R² is OH, and R³ is 4-O₂NC₆H₄.